Expression Validation by qRT-PCR
To validate and check the reliability of RNA-Seq data, qRT-PCR analysis
was performed. Twelve stress induced genes were selected for
experimental validation by qRT-PCR. From each biological replicate, cDNA
was synthesized using the HiScriptII-QRT-superMix through qPCR+ gDNA
wiper (Vazyme, Nanjing, China) in accordance with manufacturer’s
instruction. ABI 7500 Real-Time PCR System (Applied Biosystems, USA) was
used for qRT-PCR analysis. Each reaction comprises of 10µl 2×SYBR Green
Master Mix (Applied Biosystems, USA), 2.0µl cDNA, and 400 nM primer
(forward, reverse) upto final volume of 20µl. The PCR conditions were
95˚C for 3 min followed by 40 cycles of 95˚C for 10 s, 60˚C for 30 s and
65˚C for 5s. Moreover, expression level were normalized by using actin
gene (GmActin11 , as an internal control) and Ct method (Livak and
Schmittgen, 2001). For each sample, qRT-PCR reaction was repeated in two
replicates, and three technical repeats of each biological replicate.
The PRIMER5.0 software was used to design primers, and all primers are
listed in Supplementary Table S1.